Project Summary/Abstract In substantial contrast to conventional cigarettes, battery-operated electronic cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of nicotine, flavoring chemicals and humectants. According to the Centers for Disease Control and Prevention, in 2014 more than 9 million Americans used e-cig on a regular basis. Little, however, is known regarding the long-term pulmonary effects of inhaling e- cig aerosolized nicotine and flavors. Heating of e-liquids during e-cig use produces an aerosol that includes fine and ultrafine particles, as well as nicotine and aldehydes, which can produce declines in lung function, damage to epithelial cells, and induce lung inflammation. E-cig users inhale these aerosols and control the choice of e- liquids and design features (resistance and voltage) of their third-generation (3rd-Gen) e-cig devices. These are key factors that can significantly impact the toxicity of the inhaled aerosols. Therefore, our central hypothesis is that the constituent levels and toxicity of the e-cig aerosol will change depending on the specific composition of the e-liquid, the atomizer?s resistance and the voltage applied to the e-cig device. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. Our approach will overcome current research limitations by defining the impact of 3rd-Gen e-cig design features, using standard resistance and voltage values, on inhaled e-cig aerosol pulmonary toxicity in vitro and in vivo. To date, there are no studies investigating the impact of 3rd-Gen e-cig device design features on pulmonary toxicity in both in vitro and in vivo models simultaneously. Our preliminary data demonstrate that we have optimized and characterized an innovative e-cig aerosol exposure system for both in vitro and in vivo pulmonary toxicity studies that is representative of human vaping topography and pulmonary deposition. It will enable us to obtain unique comprehensive data on e-cig aerosol exposure-pulmonary effect continuums. It will also bridge the research gap that correlates in vitro to in vivo toxicity by identifying early and sensitive pulmonary biomarkers of toxicity that can serve as tools to prevent lung disease development associated with long-term e-cig aerosol exposures. In this study, our goals are A) to examine how two design characteristics of 3rd-Gen tank-style e-cig devices - atomizer resistance and battery voltage - affect e-cig aerosol composition and cellular toxicity in vitro. This will evaluate the e-cig device atomizer and battery, as well as flavoring chemicals, as items potentially requiring regulations. B) To compare the in vitro and in vivo pulmonary toxicity responses of these generated aerosols by screening for biomarkers of pulmonary toxicity in young adult mice exposed sub-chronically by inhalation to e-cig aerosols, and in neonatal mice exposed in utero. This will help better inform the policymakers, healthcare providers and the e-cig users about the pulmonary toxicity associated with long-term e-cig use. Overall, this study will help lay the groundwork for evidence-based regulatory decisions.